Qcl 1000 endpoint chromogenic lal assay

Human cell-free Hb was purified as previously described [25 (link)] from human adult red blood cells obtained from the blood center in Lund (Sweden). The Hb concentration was quantified using Plasma/Low Hb (Hemocue, Ängelholm, Sweden). The Hb was dissolved in Ringer’s Acetate (Baxter, Deerfield, Ill, USA) and purified from endotoxin contamination using EndoTrap as described by the manufacturer (Hyglos GmbH, Germany). The absolute purity of Hb from contamination with endotoxin was determined by QCL-1000™ Endpoint Chromogenic LAL Assay as described by the manufacturer (Lonza, Switzerland).

Extracts were prepared as described previously [4 ]. Briefly, fresh blue swimmer crab (Portunus pelagicus), black tiger prawn (Penaeus monodon), banana prawn (Fenneropenaeus merguiensis/indicus) and mud crab (Scylla serrata) were purchased from local markets. For raw blue swimmer crab (RC1), raw mud crab (RC2), raw black tiger prawn (RP1) and raw banana prawn (RP2) extracts, the outer shell was removed and muscle collected. For cooked blue swimmer crab (CC1), cooked mud crab (CC2), cooked black tiger prawn (CP1) and cooked banana prawn (CP2) extracts, the outer shell was retained during the heating process (20 minutes immersed in boiling PBS) before its removal and muscle tissue extracted. Finely cut muscle was blended in PBS pH 7.2 and incubated overnight at 4°C with constant mixing. After centrifugation at 13,000 rpm at 4°C for 20 minutes the supernatant was collected, dialyzed against PBS and filter sterilized before storage at -80°C in aliquots. Extract protein concentrations were determined using the Bradford assay kit (Bio-Rad Laboratories, Hercules, CA) with bovine gamma globulin as a standard. All extracts were confirmed to be neither mitogenic nor toxic as described previously [22 (link)]. Endotoxin levels in all extracts were negligible (<2 EU/mL; QCL-1000 Endpoint Chromogenic LAL Assay; Lonza, Basel, Switzerland).

Fetal Hb was purified as previously described [21 (link)] from freshly drawn human umbilical cord blood. MetHb was prepared by incubating the purified Hb solution at 37°C for 72 hours. The metHb concentration was quantified as described previously [21 (link)]. Heme (ferriprotoporphyrin IX chloride) was purchased from Porphyrin Products Inc. (Logan, UT, USA), and a 10 mM stock solution was prepared using dimethyl sulfoxide (DMSO; Sigma-Aldrich, St. Louis, MO, USA). The metHb was purified from endotoxin contamination using the endotoxin removing product EndoTrap (Hyglos GmbH,Bernried am Starnberger See, Germany) as described by the manufacturer. The absolute purity of metHb and heme from contamination with endotoxin (0 EU/mg Hb/heme) was determined using the QCL-1000™ Endpoint Chromogenic LAL Assay (Lonza, Switzerland) as described by the manufacturer.

PPE implants (MEDPOR, Stryker Craniomaxillofacial, Portage, MI, USA) with a pore size of 100–200 µm were cut to a scaffold size of 50 mm × 3 mm × 0.1 mm under sterile conditions and again sterilized upon implantation by steam sterilization (5050 ELVD, Tuttnauer, Breda, Netherlands) upon further processing. After steam sterilization the implants showed no altered structure and sample implants were negatively tested for endotoxin contamination (QCL-1000TM Endpoint Chromogenic LAL Assay, Lonza Group, Basel, Switzerland).

PBMCs were isolated by standard density gradient centrifugation with Ficoll Paque Plus (GE Healthcare, 17-1440-03) from human buffy coats. Buffy coats were obtained from German Red Cross (Berlin, Germany) according to the current version of the declaration of Helsinki with an approved ethic vote (Charité, Berlin Germany; EA4/071/13). Untouched CD14⁺ and CD14⁺CD16⁺ monocytes were isolated by negative depletion using the human PAN monocyte isolation kit (Biolegend, 480060), following the manufacturer's instructions. Monocytes were cultured in commercial ready-to-use MoDC differentiation medium containing 400 IU/ml IL-4 and 500 IU/ml GM-CSF (Miltenyi Biotec, 130-094-812) at 37°C with 5% CO₂. Every two days, half of the culture medium was replaced with fresh medium. On day six, immature MoDCs were harvested, washed with PBS and resuspended in fresh medium at a density of 10⁶ cells/ml. Cells were either treated with medium only as control, 2.5 μg/ml LPS (from Escherichia coli O111:B4, L3024-5MG) or 400 μM NiSO4 (31483, tested endotoxin-free using QCL-1000TM Endpoint Chromogenic LAL Assay (Lonza, 50-647U) according to the manufacturers protocol). For proteomic studies and flow cytometry, cells were harvested after 24 h of continuous chemical incubation; for quantitative RT-PCR, cells were incubated either for 24 h or over a 36 h period with sample collections at various time points.